Cellular insights into reactive oxidative species (ROS) and bacterial stress responses induced by antimicrobial blue...

The global threat of antimicrobial resistance (AMR), largely driven by the long-term misuse of antibiotics, highlights the urgent need for decentralized disinfection methods. Our previous study confirmed the effectiveness of antimicrobial blue light (aBL) against AMR. However, the underlying cellular mechanisms remained unexplored. However, the underlying cellular mechanisms remained unexplored. Here, we address this gap by combining selective ROS scavenger assays with transcriptional profiling of oxidative stress responsive genes, thereby providing mechanistic insights into the inactivation of facultative pathogenic bacteria (FPB) and antibiotic resistance genes (ARGs) under aBL treatments. A multidrug-resistant Pseudomonas aeruginosa and vanA-positive Enterococcus faecium reference strain were treated with aBL alone and aBL combined with TMPyP. Discrepancies between cultivation and quantitative PCR (qPCR) results revealed the presence of viable but non-culturable (VBNC) cells in case of P. aeruginosa, underscoring the necessity of combining both approaches to reliably assess aBL inactivation. Among all treatments, aBL combined with sub-lethal TMPyP concentrations of (10^-6 M) proved the most effective, with reactive oxidative species (ROS) analysis identifying singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂) as the primary reactive species responsible for bacterial inactivation. Stress responsive gene expression analysis under three treatment conditions (aBL alone, aBL + H₂O₂ (1 mM), aBL + TMPyP (10^-6 M)) showed an upregulation of sodA (>2.4-fold), recA (>2.5-fold), oxyR (>3.1-fold), and tolC (>4.2-fold), particularly under aBL + TMPyP treatment, indicating strong oxidative stress responses. In contrast, the downregulation of ompF suggests a membrane-protective mechanism aimed at reducing permeability to harmful substances. Unlike previous aBL studies, our work links ROS specificity with bacterial stress adaptation, supporting the potential of aBL as a scalable and mechanistically understood strategy for decentralized wastewater disinfection at AMR hotspots.